Oligonucleotide linkers and primers have been used in the prior art for priming, binding or annealing single strand polynucleotides and allowing the synthesis of the second polynucleotide complementary strand.
All of Carninci et al., 1996, Genomics, 37, 327-336; Carninci et al., 1997, DNA Research 4:61-66; Carninci et al., 1998, Proc. Natl. Acad. Sci USA, 95:520-4; Carninci and Hayashizaki, 1999, Methods Enzymol. 303:19-44, disclose methods for the preparation of cDNA libraries. According to these protocols, a mRNA/cDNA hybrid is prepared and full-coding/full-length cDNAs are selected by mean of the Cap trapper technology, then each single strand cDNA is ligated with a G-tail and the cDNA second strand is synthesized.
However, the G-tailing methodology shows several drawbacks, for example, in sequencing efficiency and translation efficiency when cDNAs clones are used for protein expression.
G-tailing is performed by terminal addition of dGTP using terminal deoxynucleotidyl transferase. However, the number of G residues added is difficult to control and it is variable, generally between 10 and 30. A long G-tail has the drawback of impairing a long read sequencing and lowering the sequencing efficiency, whilst a short G-tail has the drawback of providing a low efficient priming, with the consequence of loss of sample, and necessity of repreparing it.
During a sequencing reaction, long G-stretches (long G-tail) interact with surrounding sequences and form very strong secondary structures. This may be problematic in case of interactions with 5′ UTRs that are typically GC rich. In fact, a typical cDNA has 60% GC content in the 5′-UTR that is considered to act as a regulatory region. Similar problems were also observed in cloning vectors having GC rich regions containing a Sfi I or Not I restriction site next to the cloning site.
Further, terminal deoxynucleotidyl transferase used for tailing reaction requires heavy metals, for example, MnCl2 or CoCl2. However, these heavy metals have sometimes caused degradation of cDNAs and decreased rate of production of long strand, full-coding/full-length cDNA.
The purpose of the present invention is to solve these several problems in the prior art and provide a novel and efficient method for the preparation of cDNA libraries.
More specifically, the purpose of the present invention is to provide a novel linker, which can be utilized instead of G-tailing in a method for the preparation of cDNA libraries and to provide a method for the preparation of cDNA libraries using said linker.